One fifth of global mortality has been reported to be due to infectious disease. Within this, respiratory diseases alone account for about four million deaths per year (Girard et al. (2005) Vaccine 23, 5708-24). However, because of the expense of development, coupled with uncertainty regarding efficacy, only about 2% of the global pharmaceutical budget is allocated to development of new prophylactic vaccines (Kiney & Girard (2005) Vaccine 23, 5705-7). Given the scope of the worldwide health problems caused by known and emerging infectious diseases, and additionally, the potential of novel biological warfare pathogens, it is important that novel strategies of rapid vaccine evaluation be developed and implemented.
Lung Structure and Function
The mammalian lung is an organ within the thorax where passive diffusion of oxygen and carbon dioxide occur across a thin, two-cell-thick tissue. Normal lung function is to exchange oxygen from the air with carbon dioxide in the blood. Only two cell layers are interposed between the air and the blood, with a small amount of ECM serving as a scaffold.
The respiratory mucosa is the largest organ system directly open to the outside environment, with a surface area of 60-80 m2. It serves as a point of entry for nutrient gases, along with microorganisms and particulate antigens that can trigger a multitude of respiratory immune responses. The first and the last point of contact occur at the nasopharynx and the alveolar regions of the lung, respectively.
The alveolus is the site where gaseous exchange occurs between the alveolar type I cells (respiratory epithelial cells), alveolar type II cells, and the vascular endothelial cells of the surrounding capillaries. These two cell layers prevent the direct mixing of air and blood, yet provide throughout the lung alveoli a massive surface area for gaseous exchange to occur. In an adult human, this surface area is about 60 m2 during full expiration and about 80 m2 during full inspiration.
Many pathophysiological conditions result in the direct increase or reduction of pulmonary mass, leading to a decrease in gaseous exchange. Non-malignant and malignant primary and metastatic lung tumors are principal reasons for permanent decreases in pulmonary function. Other causes of decreased pulmonary function include viral and bacterial pneumonias, trauma, fibrosis, and idiopathic disease.
The alveoli are tiny air sacs, the walls of which are covered with capillaries across which oxygen and carbon dioxide readily diffuse and are transferred into and out from the blood, respectively. This exchange is essential to survival and is the key function of the lungs.
The alveolus is a site where only two cell layers are interposed between the air and the blood, with a small amount of ECM serving as a membranous scaffold. Gaseous exchange occurs across the alveolar wall, comprising alveolar type I and type II cells (squamous respiratory epithelial cells) and the vascular endothelial cells of the surrounding capillaries.
Alveoli have fragile, thin walls, which are easily damaged. Breakage of these walls makes the oxygen-carbon dioxide diffusion much less efficient. The bronchial tree distributes the air throughout the lung to the individual alveoli. Once damaged, the bronchioles tend to collapse, trapping stale air in the isolated sacs and not letting fresh air in, leading to atelectasis.
Emphysema permanently enlarges and irreversibly damages the alveoli. It damages the ends and walls of the smallest bronchioles (the tiny breathing tubes that branch off from the trachea and bronchi), and diminishes pulmonary elasticity.
As alveoli and bronchial tubes are destroyed in pathophysiological conditions, progressively more air is required to provide the same amount of oxygen to the blood via the parts of the lung that are still functioning. This need for more air eventually leads to lung over-inflation. As the lung over-expands, it gradually enlarges, completely filling the maximum thoracic cage volume and causing a sense of shortness of breath. Because the lung can no longer expand or contract as completely as before, stale air left in the lung is never completely replaced with fresh air, resulting in poor gas exchange. The combination of a larger, less elastic lung and damaged, non-functioning tissue means that the air flow out of the lung is much slower, resulting in the feeling of an obstructed airway.
Many lung diseases that cause a narrowing of the respiratory airways (e.g., chronic bronchitis, asthma) can contribute to the onset of emphysema, but smoking is a common cause. In addition to the irreversible damage smoking causes to lung tissue, it causes inflammation of the lungs, which resolves only when smoking stops. Smoking also stresses the natural antioxidant defense system of the lung, allowing free radicals to damage lung tissue at the cellular level.
Additionally, irritants contained in tobacco smoke tend to inhibit activity of the cilia of the airways. These cilia ordinarily function to expel foreign matter and mucus from the lung. Without their activity, it becomes difficult or impossible to cough up the mucus that accompanies pneumonia and other lung infections. Cigarette smoke can temporarily paralyze the cilia. Smoking-induced emphysema usually becomes apparent after age 50.
The deposition of an inhaled particle in the lung has been linked to its size. For example, the upper respiratory mucosa is the first anatomic barrier where particles from ˜5 to ˜10 μm are deposited, while ˜0.2 to ˜2-μm sized particles are deposited in the lower mucosal alveolar region.
At the molecular level, the initial responses to such foreign particles include opsonizing agents (e.g., collectins), activation of cytochrome P450s, complement, lysozymes, anti-bacterial peptides (e.g., defensins), mannose-binding proteins, and interferons. Phagocytic cells activated at primary antigen deposition sites include alveolar macrophages and natural killer (NK) cells, which have the ability to recognize and neutralize infected cells, through recognition of bacterial and viral features. The adaptive responses trigger T and B lymphocytes that build immunological memory to subsequent challenges. This process is primarily regulated by antigen-presenting cells (APCs), such as macrophages, dendritic cells (DCs), and Type II epithelial cells. The T cell receptor (TCR) on the surface of the T lymphocyte is only activated by sensing major histocompatibility complex (MHC) molecules containing processed antigenic peptides on the surface of APCs. T cell responses are dependent on cytokines produced and functional effects after encountering antigen-specific T cells.
Respiratory epithelial cells have been shown to be responsive to lipopolysaccharides (Koyama et al. (1991) J. Immunol. 147, 4293-301; Diamond & Bevins (1994) Chest 105(3 Suppl), 51S-52S), muramyl dipeptides (Lopez-Boado et al. (2000) J. Cell Biol. 148, 1305-15; Diamond et al. (2000) Infect Immun. 2000 January; 68(1):113-9; Bevins (2003) Contrib. Microbiol. 10, 106-48), and lipoteichoic syncytial acid (Wagner et al. (1999) Am. J. Respir. Cell Mol. Biol. 20, 769-76; Diamond et al. (2000) Infect Immun. 2000 January; 68(1):113-9). They express Toll-like receptors (Holgate (2007) Trends Immunol. 28, 248-51; Bals & Hiemstra (2004) Eur. Respir. J. 23, 327-33; Becker et al. (2000) J. Biol. Chem. 275, 29731-6), TNF receptors (Levine (1995) J. Investig. Med. 43, 241-9; Nettesheim & Bader (1996) Toxicol. Lett. 88, 35-7), and demonstrate up-regulation of host defense genes, such as like MUC2, MUC5C, hBD2, and LL37/CAP18 (Becker et al. (2000) J. Biol. Chem. 275, 29731-6; Agerberth et al. (1999) Am. J. Respir. Crit. Care. Med. 160, 283-90; Bartman et al. (1998) J. Pathol. 186, 398-405; Yoon & Park (1998) Rhinology 36, 146-52; Dohrman et al. (1998) Biochim. Biophys. Acta 1406, 251-9; Li et al. (1997a) J. Pathol. 181, 305-10; Li et al. (1997b) Proc. Natl. Acad. Sci. USA 94, 967-72). Respiratory epithelium also produces interleukins (IL-1, IL-5, IL-6, IL-8), RANTES (regulated upon activation, normal T cell-expressed, and secreted), endothelin, granulocyte-monocyte colony stimulating factor (GM-CSF), transforming growth factor beta (TGF-β), interferon-γ-induced protein (IP-10), interferon-inducible T-cell α-chemoattractant (I-TAC), and γ-interferon-inducible T cell chemoattractant (Chung (2006) Curr. Drug Targets 7, 675-81; Prescott (2003) J. Paediatr. Child Health 39, 575-9; Holt & Stumbles (2000) J. Allergy Clin. Immunol. 105, 421-429).
As part of the adaptive immune response, memory T cells are constantly circulating through the lung parenchyma including alveolar spaces via well characterized lymphocyte homing mechanisms (Wardlaw et al. (2008) Clin. Exp. Allergy 35, 4-7). As the lung is considered a tertiary lymphoid organ (Grigg & Riedler (2000) Am. J. Respir. Crit. Care 162, 52-5), it contains large numbers of memory T cells in all compartments of the respiratory tract, with the largest number in the lung, migrating in through the post capillary venules under low hydrodynamic pressures. These memory T cells migrate specifically to the organ of their cognate antigen initiation. Much fewer naïve T cells enter the lung as age increases and only respond to newly seen antigens. These naïve T cells must enter the lung alveolar parenchyma via high endothelial venules under higher vascular pressures and faster flow. Unregulated T cell emigration into alveolar spaces and respiratory parenchyma may be a key factor for the formation of asthma (Bedoff et al. (2008) Annu. Rev. Immunol. 26, 205-32).
Respiratory Delivery Route
Advances in respiratory mucosal delivery have been driven by the non-invasive, highly absorptive properties of the respiratory route. Compared with the oral route, respiratory delivery offers a lack of digestive enzymes or mechanical forces, along with thin walls and a highly absorptive vascularized surface area for improved systemic delivery.
For vaccine or drug delivery, nebulizers and powder inhalers allow deposition of therapeutics in specific sites of the lung (Byron (2004) Proc. Am. Thorac. Soc. 1, 321-8; Laube (2005) Respir. Care 50, 1161-76). The potential for aerosolized vaccine delivery for influenza and measles, in addition to delivery of peptides and small molecule drugs has been explored with some success in asthma, chronic obstructive pulmonary disease (COPD), migraine, and diabetes-related therapeutics (Laube (2005) Respir. Care 50, 1161-76; Kennedy (1991) Drugs 42, 213-27; Illum (2002) Drug Discov. Today 7, 1184-9; Sullivan et al. (2006) Expert Opin. Drug Deliv. 3, 87-95). A prominent FDA-approved aerosolized vaccine is FluMist® vaccine, for seasonal flu. However, our limited understanding of the mechanistic details of the respiratory drug delivery has hampered the development of aerosolized therapeutics.
Other Respiratory Immunology/Toxicology Models: In Vivo and In Vitro Approaches
Since the 1970s there has been interest in developing in vivo respiratory models to study human immunology (e.g., Chowhan & Amaro (1976) J. Pharm. Sci. 65, 1669-72; Torkelson et al. (1976) Am. Ind. Hyg. Assoc. J. 37, 697-705; Belshe et al. (1977) J. Med. Virol. 1, 157-62; Saffiotti (1978) Environ. Health Perspect. 22, 107-13; Schanker (1978) Biochem. Pharmacol. 27, 381-5). Previous in vitro lung models included those based on the Transwell™ cell culture permeable support device or similar construct, comprising an endothelial cell layer, an epithelial cell layer, and an artificial microporous membrane, having pores therein, disposed between and in direct contact with the endothelial cell layer and the epithelial cell layer such that the membrane has an endothelial side and an epithelial side (see, e.g., U.S. Pat. No. 5,750,329; Weppler et al. (2006) Exp. Lung Res. 32, 455-82; Birkness et al. (1995) Infect. Immun. 63, 402-9; Birkness et al. (1999) Infect. Immun. 67, 653-658).
U.S. Pat. No. 5,750,329 describes a method for constructing an artificial lung system, comprising placing an artificial microporous membrane, having pores therein, into a vessel having a bottom and supporting the membrane a distance from the bottom of the vessel to create an upper and lower chamber in the vessel such that the membrane has an endothelial side facing into the lower chamber of the vessel and an opposite epithelial side facing into the upper chamber of the vessel; placing endothelial cells into the upper chamber of the vessel under conditions such that the endothelial cells form a confluent layer of cells on the epithelial side of the membrane; and placing alveolar epithelial cells into the upper chamber of the vessel under conditions such that the endothelial cells migrate through the pores in the membrane and attach to the endothelial side of the membrane to form a confluent layer of the endothelial cells on the endothelial side of the membrane in the lower chamber and the alveolar epithelial cells form a confluent layer of the epithelial cells on the epithelial side of the membrane in the upper chamber.
While in vivo animal models have been developed, variations in animal size, species, and differences in therapeutic distribution have resulted in inconsistencies in reported findings by various research groups using such models. Most in vivo models require destructive means for animal dosing using test compounds and vaccines, and blood and tissue isolation that also depend on animal handling/surgical protocols that are under scrutiny (Schanker & Hemberger (1984) Pharmacology 28, 47-50; Schanker & Hemberger (1983) Biochem. Pharmacol. 32, 2599-601; Schanker et al. (1986a) Pharmacology 32, 176-80; Schanker et al. (1986b) Drug Metab. Dispos. 14, 79-88; Schanker (1978) Biochem. Pharmacol. 27, 381-5; Hemberger & Schanker (1983a) Drug Metab. Dispos. 11, 73-4; Hemberger & Schanker (1983b) Drug Metab. Dispos. 11, 615-6; Brown & Schanker (1983a) Drug Metab. Dispos. 11, 355-60; Brown & Schanker L S (1983b) Drug Metab. Dispos. 11, 392-3; Lin & Schanker (1983a) Drug Metab. Dispos. 11, 75-6; Lin & Schanker (1983b) Drug Metab. Dispos. 11, 273-4; Mobley & Hochhaus (2001) Drug Discov. Today 6, 367-375, Widdicombe (1997) J. Appl. Physiol. 82, 3-12; Flecknell (2002) ALTER 19, 73-8; Abbott (2005 Nature 438, 144-6). Generally, the cost and time required for in vivo animal models along with physiological variations in the selected animal and human species (Li et al. (2007) Exp. Lung Res. 33, 227-44; Cao et al. (2007) Toxicol. Lett. 171, 126-37; Denham et al. (2007) Am. J. Physiol. Lung Cell. Mol. Physiol. 292, L1241-7; Carter et al. (2006) J. Occup. Environ. Med. 48, 1265-78) makes them less attractive than in vitro models for many purposes.
Compared with in vivo approaches, in vitro models are considered more humane, cost effective, are generally simpler to execute, and can use more physiologically relevant human cells. Various approaches have been adopted to develop in vitro respiratory system models using epithelial cell lines. However, the intrinsic differences between the functionality of these cell lines and native primary cells make these systems less than ideal. Several lung cell lines have been reported, including carcinoma-derived epithelial cell lines, such as A549 (alveolar), Calu-1, Calu-3, Calu-6, H441, HBE1, and A427. Normal tissue-derived transformed cell lines include 16HBE14o- (bronchial), 9HTE16o- (tracheal), 1HAEo- (tracheobronchial), BEAS-2B (bronchial), and CF/T43 (nasal). However, undesirable changes in cell line characteristics over passage culture have been reported, such as morphology, growth rates, protein expression, permeability, and signaling (ATCC Technical Bulletin no. 7, Passage number effects on cell lines. 2007 1-3; Esquenet et al. (1997) J. Steroid Biochem. Mol. Biol. 62, 391-9; Briske-Anderson et al. (1997) Proc. Soc. Exp. Biol. Med. 214, 248-57; Chang-Liu & Woloschak (1997) Cancer Lett. 113, 77-86; Yu et al. (1997) Pharm. Res. 14, 757-62, Sambuy et al. (2005) Cell Biol. Toxicol. 21, 1-26; Wenger et al. (2004) Biosci. Rep. 24, 631-9).
Such intrinsic differences can also be of concern when developing in vitro screening systems for therapeutics and pathogens. The need for highly functional primary lung epithelial cells has led to development of a number of primary cell isolation methods for mouse (Corti et al. (1996)Am. J. Respir. Cell Mol. Biol. 14, 309-15), rat (Goodman & Crandall (1982) Am. J. Physiol. 243, C96-100; Cheek et al. (1989) Exp. Cell Res. 184, 375-87) rabbit (Shen et al. (1999) Pharm. Res. 16, 1280-7), pig (Steimer et al. (2006) Pharm. Res. 23, 2078-93), and human lung tissue (Bur et al. (2006) Eur. J. Pharm. Sci. 28, 196-203; Elbert et al. (1999) Pharm. Res. 16, 601-8). However, differences between tissue responses between species have been noted, a possible drawback to using primary animal lung cells (Denham et al. (2007) Am. J. Physiol. Lung Cell. Mol. Physiol. 292, L1241-7; Carter et al. (2006) J. Occup. Environ. Med. 48, 1265-78).
Viral pathogens that cause respiratory disease include common flu or influenza (A or B; Orthomyxoviridae family), respiratory syncytial virus, human parainfluenza viruses (HPIVs; paramyxovirus family), metapneumovirus (hMPV; family Paramyxoviridae), adenoviruses, rhinoviruses, parainfluenza viruses, coronaviruses, coxsackievirus, and herpes simplex virus. Respiratory disease bacterial pathogens include Yersinia pestis, Bacillus anthracis, Escherichia coli, Francisella tularensis, Staphylococcus aureus Group A beta-hemolytic streptococci (GABHS), group C beta-hemolytic streptococci, Corynebacterium diphtherias, Neisseria gonorrhoeae, Arcanobacterium haemolyticum, Chlamydia pneumoniae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Bordetella pertussis, and Bordetella parapertussis. 
There is a continuing need for a predictive, reproducible in vitro model system based on lung immunophysiology and function that would enable the study of a broad spectrum of respiratory disease pathogenesis and associated therapies. There is also a continuing need for in vitro immunological approaches for accurately predicting human immunological responses. The artificial tissue constructs of the present invention, with their use of three-dimensional (3D) tissue engineering and advanced cell biology, combined with modern bio-fabrication and bioreactor techniques, provide such a model system.